树鼩脑缺血时海马微环境与血脑屏障通透性改变的可能机制

目的观察树鼩血栓性脑缺血时海马血脑屏障（blood-brain barrier，BBB）通透性的变化，并探讨其与微环境相互作用的可能机制。方法通过光化学反应诱导树鼩血栓性脑缺血模型，采用单泵等速微灌流系统及荧光分光法检测脑缺血后4、24及72h海马细胞外液异硫氰基荧光素标记的葡聚糖（fluorescein thiocarbamoyl dextrans，FITC-D）含量及血浆FITC-D的清除率（Clearancerate of FITC-D，Cf），Cf的高低反映海马BBB的通透性。用高效液相色谱技术检测海马灌流液的氨基酸递质水平，并观察缺血海马微血管超微结构。结果树嗣血栓性脑缺血后4h海马BBB通透性开始升高，缺血24h最为明显，Cf=（0．526±0．130）／μl（P〈0．01），72h通透性有所恢复。脑缺血后24h海马细胞外Glu及GABA水平明显升高，分别为（5．71±0．39）μmol·L^-1和（3．81±0．14）μmol·L^-1，均具有统计学意义（P〈0．01）。海马微血管超微结构观察显示星形胶质细胞肿胀，毛细血管周围水肿形成。结论脑缺血可导致海马BBB通透性增高，而海马微环境紊乱对BBB通透性具有促进作用。     

缺血后适应调控树鼩脑缺血海马CA1区Akt（pS473）和Akt（pT308）过磷酸化的抗凋亡机制

观察树鼩脑缺血海马神经元Akt（pS473）和Akt（pT308）磷酸化改变对CA1区细胞凋亡的影响,探讨缺血后适应（PC）抑制细胞凋亡的可能机制。方法 用光化学诱导法诱导树鼩脑缺血并建立缺血PC模型;用免疫组织化学TACS原位凋亡检测试剂盒检测皮层及海马CA1区神经元凋亡数量,用免疫组织化学法检测Akt（pS473）和Akt（pT308）表达的空间分布,用ELISA法检测海马CA1区神经元Akt（pS473）和Akt（pT308）磷酸化水平;用电子显微镜观察其神经元超微结构改变。 结果 脑缺血后神经元皱缩,核消失以缺血24h为著。脑缺血后4 h、24 h及72 h皮层及海马CA1区神经元TUNEL阳性细胞数明显增加（P<0.01）,海马CA1区神经元Akt (pS473) 和Akt (pT308)的表达及磷酸化水平明显升高,以脑缺血4 h的改变最明显,分别为(152.3±3.5) units/mg、(130.8±2.6) units/mg和(149.5±4.7)units/mg和 (42.35±2.49) units/mg、(19.23±1.41) units/mg和(23.38±1.32) units/mg (P<0.01）。缺血PC组神经元损伤明显减轻,皮层及海马CA1区TUNEL阳性细胞明显减少（P<0.01）,且与海马神经元Akt (pT308)活化水平呈平行改变(P<0.05）。结论 树鼩脑缺血海马CA1区细胞凋亡与Akt(pS473) 和Akt (pT308)过磷酸化的信号机制有关,缺血PC抑制海马神经元Akt(pS473) 和Akt(pT308)双磷酸化可能具有抗凋亡作用。     

高血糖对树鼩脑皮层局灶性缺血时海马微环境离子稳态及神经元继发性损伤的影响

 目的：研究高血糖对树鼩脑皮层血栓性缺血时海马微环境离子稳态的影响,探讨高血糖在缺血后神经元继发性损伤中的作用及机制。方法：用链脲佐菌素复制树鼩高血糖模型,并用光化学方法诱导脑皮层局部血栓性缺血,用单泵等速微灌流系统和离子分析仪测定缺血4 h、24 h及72 h海马离子微环境（细胞外pH值、K+、Na+、Ca2+、Cl-）的动态变化,并观察脑组织的病理形态学改变及海马神经元密度变化。结果：树鼩脑皮层缺血后,其海马微环境内出现了pH值、Na+、Ca2+及Cl-含量的降低,K+含量升高,变化以缺血后4 h为著,24 h次之,72 h无显著差异;高血糖加缺血进一步加重离子稳态的紊乱,缺血后4 h的pH值、K+和Ca2+含量,以及缺血后24 h的pH值和Na+含量与正常血糖缺血组同期值相比,变化显著（P<0.05）。形态学观察显示,光化学反应后4 h照射区皮层可见梗死灶,且患侧海马CA1区也存在缺血损伤性改变;24 h病损达高峰;72 h伴随胶质细胞增生等修复性反应。相应时点高血糖加缺血组皮层及海马的损伤均大于缺血组,以缺血后24 h(P<0.01)和72 h(P<0.05)尤为显著。结论: 树鼩脑皮层血栓性缺血形成后,缺血中心区扩布所导致的微环境内酸碱平衡及离子稳态性异常可能是海马神经元继发性损伤的重要原因,高血糖可加剧缺血脑区离子微环境的紊乱。     

缺血后适应减轻树鼩缺血性脑水肿及脑梗死的机制

目的 观察缺血后适应对树鼩血栓性脑缺血时大脑皮层脑水含量、局部脑血流、梗塞面积及神经元超微结构的影响,探讨其对树鼩脑缺血时神经保护的可能机制。 方法 将88只健康成年树鼩随机分为对照组、脑缺血4 h组、脑缺血24 h组、后适应4 h组及后适应24 h组（每组n=8）,另取8只动物做HE染色（n=3）及电子显微镜观察（n=5）。本实验采用光化学反应诱导树鼩血栓性脑缺血而建立脑缺血动物模型,在脑缺血模型建成后4 h夹闭缺血侧颈总动脉5 min,再灌注5 min,如此交替进行3个循环以建立缺血后适应模型。测定大脑皮层局部脑血流,脑组织含水量,脑梗死范围,并观察皮层及海马CA1区神经元超微结构改变。 结果 脑缺血时神经元固缩,线粒体肿胀,嵴溶解或形成空泡,内质网肿胀,内质网池形成。缺血后适应能使海马CA1区神经元固缩减少,线粒体和内质网的病理改变减轻,细胞水肿改善。随着缺血时间的延长,缺血24h组脑水含量明显增加86.81%±1.08%,此时脑梗塞面积明显扩大33.00%±3.03%,局部脑血流明显降低（134.27±28.75）ml/min。缺血后适应24h组脑组织含水量明显减少（81.04%±1.04%,P＜0.01）;脑梗塞面积缩小（16.79%±1.29%,P＜0.01）;而局部脑血流明显增加［（195.25±21.18）ml/min,P＜0.01］。 结论 缺血后适应可缓解树鼩缺血性脑水肿并缩小梗死范围,其机制可能与改善局部脑血流有关。     

缺血后适应对树鼩脑缺血信号转导及转录激活因子3过磷酸化的调控作用

目的观察信号转导及转录激活因子3(STAT3)磷酸化在树鼩脑缺血后适应(PC)神经保护中的作用,并探讨其可能机制。方法将50只健康成年树鼩随机分为对照组、脑缺血4 h组、脑缺血24 h组、后适应4 h组和后适应24 h组(每组n=5),其中10只动物做HE染色(n=5)及电子显微镜观察(n=5)。本实验通过光化学反应建立树鼩血栓性脑缺血模型;于缺血后4 h夹闭患侧颈总动脉3次(每次5 min)实施缺血PC。采用TTC染色观察树鼩脑梗死面积的变化,通过HE和电子显微镜观察脑皮质和海马组织学改变及其超微结构变化,应用Western blotting检测皮层总STAT3(t-STAT3)及磷酸化STAT3(p-STAT3)蛋白表达变化。结果脑缺血后皮层血管内皮细胞肿胀,皮层及海马神经元损伤,线粒体肿胀、嵴溶解,以缺血24 h损伤最为明显,脑梗死面积达到(24.78±2.06)%。而皮层p-STAT3蛋白表达随缺血时间延长呈增高趋势,缺血4 h p-STAT3蛋白表达明显增高(0.24±0.1,P<0.01),缺血24 h p-STAT3蛋白表达则持续增高(0.32±0.1,P<0.01)。缺血PC处理后皮层血管内皮细胞水肿好转,皮层及海马神经元损伤减轻,脑梗死面积减小为(17.67±1.90)%(P<0.01)。与缺血组相比,缺血PC 4 h p-STAT3蛋白表达进一步升高(0.41±0.09,P<0.01),缺血PC 24 h p-STAT3蛋白表达增高更加显著(0.70±0.11,P<0.01)。结论树鼩脑缺血可导致STAT3磷酸化代偿性增强,缺血PC的脑保护作用可能与其促进STAT3过磷酸化有关。     

脑缺血及后适应对树鼩海马内质网应激信号分子PERK及GRP78的影响

目的:探讨脑缺血及后适应对树鼩海马内质网应激信号分子蛋白激酶R样内质网激酶(PERK)及葡萄糖调节蛋白78(GRP78)的影响及后适应的脑保护机制。方法:光化学反应诱导树鼩局部血栓性脑缺血,于脑缺血后3.5 h夹闭、打开缺血侧颈总动脉交替3个循环(每次5 min)以复制缺血后适应模型。HE染色观察缺血侧海马神经元损伤及超微结构变化,RT-PCR检测脑缺血及后适应不同时间海马组织PERK及GRP78 mRNA表达的变化,免疫组织化学法与Western blot法检测PERK及GRP78的蛋白定位及表达变化。结果:海马神经元随脑缺血时间延长而损伤加重,缺血24 h损伤最为严重,后适应可减轻损伤。脑缺血4 h、24 h及72 h PERK的mRNA及蛋白表达较假手术组增高(P0.05),后适应组与相应的缺血组相比,PERK的mRNA及蛋白表达减少,4 h、24 h差异显著(P0.05);脑缺血4 h、24 h及72 h GRP78的mRNA及蛋白表达与假手术组无明显差异,后适应24 h组较缺血24 h组表达增高(P0.05)。结论:树鼩局部血栓性脑缺血可激活缺血侧海马内质网应激反应,引起PERK/e IF2α信号转导通路中相关分子PERK的mRNA及蛋白表达增高。缺血后适应处理通过下调PERK、上调GRP78的表达,减轻内质网应激反应,减少神经元损伤,具有一定的脑保护作用。     

Changes in the bioenergetic state of rat hippocampus during 2.5 min of ischemia, and prevention of cell damage by cyclosporin A in hyperglycemic subjects

 A recent study from this laboratory has shown that brief transient ischemia (2 min 30 s) in normo- and hyperglycemic rats leads to moderate neuronal necrosis in CA1 cells of the hippocampus, of equal density in the two groups. However, hyperglycemic animals failed to depolarize during the ischemia, nor did they show a decrease in extracellular calcium concentration. The present study was undertaken to study the metabolic correlates to these unexpected findings. Normoglycemic (plasma glucose ∼6 mM) and hyperglycemic (∼20 mM) rats were subjected to ischemic periods of 1 min and 2 min 15 s (2 min 30 s with freezing delay considered), and their brains were frozen in situ. Samples of dorsal hippocampus were dissected at –22°C and extracted for the measurement of phosphocreatine (PCr), creatine, ATP, ADP, AMP, glucose, glycogen, and lactate. Normoglycemic animals showed rapid depletion of PCr, ATP, glucose, and glycogen, and a rise in lactate content to 10–12 mM·kg^–1 during the ischemia. Hyperglycemic animals displayed a more moderate rate of fall of PCr and ATP, with ATP values exceeding 50% of control after 2 min 30 s. Glycogen stores were largely maintained, but degradation of glucose somewhat enhanced the lactic acidosis. The results demonstrate that hyperglycemic rats maintained ATP at levels sufficient to prevent cell depolarization and calcium influx during the ischemic period. However, the metabolic perturbation observed must have been responsible for the delayed neuronal damage. We speculate that lowered ATP, increased inorganic P, and oxidative stress triggered a delayed mitochondrial permeability transition (MPT), which led to delayed neuronal necrosis. This assumption was supported by a second series of experiments in which CA1 damage in hyperglycemic rats was prevented by cyclosporin A, a virtually specific inhibitor of the MPT. Received: 24 May 1996 / Accepted: 30 September 1996     

Association analysis of <Emphasis Type="Italic">SLC22A4</Emphasis>, <Emphasis Type="Italic">SLC22A5</Emphasis> and <Emphasis Type="Italic">DLG5</Emphasis> in Japanese patients with Crohn disease

Crohn disease (CD) is an inflammatory bowel disease characterized by chronic transmural, segmental, and typically granulomatous inflammation of the gut. Recently, two novel candidate gene loci associated with CD, SLC22A4 and SLC22A5 on chromosome 5 known as IBD5 and DLG5 on chromosome 10, were identified through association analysis of Caucasian CD patients. We validated these candidate genes in Japanese patients with CD and found a weak but possible association with both SLC22A4 (P=0.028) and DLG5 (P=0.023). However, the reported genetic variants that were indicated to be causative in the Caucasian population were completely absent in or were not associated with Japanese CD patients. These findings imply significant differences in genetic background with CD susceptibility among different ethnic groups and further indicate some difficulty of population-based studies.     

Patterns of C-heterochromatin and telomeric DNA in two representative groups of small apes,the genera <Emphasis Type="Italic">Hylobates</Emphasis> and <Emphasis Type="Italic">Symphalangus</Emphasis>

The course of chromosome evolution in small apes is still not clear, though painting analyses have opened the way for elucidating the puzzle. Even the C-banding pattern of the lar-group of gibbons (the genus Hylobates) is not clarified yet, although our previous studies suggested that lar-group gibbons have a unique C-banding pattern. We therefore made observations to establish C-banded karyotypes of the agile gibbons included in the lar-group. The data were compared with those of siamangs (the genus Symphalangus), which carry distinctive C-bands, to determine the chromosomal patterns in each group. C-banded chromosomes of agile gibbons showed several terminal, interstitial and paracentric bands, whose patterns are specific for each chromosome, whereas the C-bands of siamangs were located only at the terminal and centromeric regions in most chromosomes. Moreover, the C-bands of agile gibbons and siamangs were shown to be G+C-rich and A+T-rich DNA, respectively, by DAPI/C-band sequential staining. Additionally, PRINS labelling with a telomere primer revealed that agile gibbons have telomeric DNA only at chromosome ends where there is no C-band (non-telomeric heterochromatin), whereas the telomeric DNA of siamangs is located in the terminal C-banded regions (telomeric heterochromatin). Although the evolutionary mechanisms in small apes are still unknown, C-banding patterns and distribution of telomeric DNA sequences should provide valuable data to deduce the evolutionary pathways of small apes.     

A comparative study of generalized activation of the synthesis of stress proteins in adaptation to stress and hypoxia

Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 116, N ^o 8, pp. 137–139, August, 1993.     

Deposition of eosinophil-granule major basic protein and expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in the mucosa of the small intestine in infants with cow’s milk–sensitive enteropathy

Background: Cow’s milk–sensitive enteropathy (CMSE) is an important cause of chronic diarrhea and failure to thrive in infancy. The immunopathology of the mucosal lesion associated with CMSE has not yet been described. Objectives: This study investigated the eosinophil activation and the role of adhesion molecules in the pathogenesis of intestinal mucosal damage associated with CMSE. Methods: Twenty-one patients with chronic diarrhea and abnormal mucosa on duodenal biopsy specimens were included. The patients had negative responses to skin prick tests and RASTs with milk. Fourteen patients were diagnosed with CMSE by milk challenge test and were designated as the CMSE group. Seven patients with no milk intolerance were defined as the non-CMSE group. Four infants with frequent vomiting and no mucosal abnormalities were also studied as the control group. Immunohistochemical stains for eosinophil major basic protein (MBP), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 on endoscopic duodenal biopsy specimens were performed. Results: The degree of eosinophil degranulation, as evidenced by localization of extracellular MBP, was significantly greater in the CMSE group compared with the non-CMSE and control groups (P < .05). Expression of VCAM-1 on mononuclear cells was higher in the CMSE group compared with the non-CMSE and control groups (P < .05). The severity of villous atrophy was positively correlated with the deposition of MBP (r = 0.79, P < .001). Conclusion: These results strongly suggest eosinophils and VCAM-1 are implicated in the pathogenesis of mucosal damage associated with CMSE. (J Allergy Clin Immunol 1999;103:1195-201.)     

Examination of the requirement for ucp-4, a putative homolog of mammalian uncoupling proteins, for stress tolerance and longevity in C. elegans

Reactive oxygen species (ROS) are generated by mitochondrial respiration and can react with and damage cellular components. According to the free radical theory of aging, oxidative damage from mitochondrial ROS is a major cause of cellular decline during aging. Mitochondrial uncoupling proteins (UCPs) uncouple ATP production from electron transport and can be stimulated by free radicals, suggesting UCPs may perform a cytoprotective function. The nematode, Caenorhabditis elegans, contains one UCP-like protein, encoded by the ucp-4 gene. We have investigated the genetic requirement for ucp-4 in normal aging and stress resistance. Consistent with the hypothesis that ucp-4 encodes a putative uncoupling protein, animals lacking ucp-4 function contained elevated ATP levels. However, the absence of ucp-4 function did not affect adult lifespan or survival in the presence of thermal or oxidative stress. Together, these results demonstrate that ucp-4 is a negative regulator of ATP production in C. elegans, but is not required for normal lifespan.     

The de novo chromosome 16 translocations of two patients with abnormal phenotypes (mental retardation and epilepsy) disrupt the <Emphasis Type="Italic">A2BP1</Emphasis> gene

The 16p13.3 breakpoints of two de novo translocations of chromosome 16, t(1;16) and t(14;16), were shown by initial mapping studies to have physically adjacent breakpoints. The translocations were ascertained in patients with abnormal phenotypes characterized by predominant epilepsy in one patient and mental retardation in the other. Distamycin/DAPI banding showed that the chromosome 1 breakpoint of the t(1;16) was in the pericentric heterochromatin therefore restricting potential gene disruption to the 16p13.3 breakpoint. The breakpoints of the two translocations were localized to a region of 3.5 and 115 kb respectively and were approximately 900 kb apart. The mapping was confirmed by fluorescence in situ hybridization (FISH) of clones that spanned the breakpoints to metaphase spreads derived from the patients. The mapping data showed both translocations disrupted the ataxin-2-binding protein 1 (A2BP1) gene that encompasses a large genomic region of 1.7 Mb. A2BP1 encodes a protein that is known to interact with the spinocerebellar ataxia type 2 (SCA2) protein. It is proposed that disruption of the A2BP1 gene is a cause of the abnormal phenotype of the two patients. Ninety-six patients with sporadic epilepsy and 96 female patients with mental retardation were screened by SSCP for potential mutations of A2BP1. No mutations were found, suggesting that disruption of the A2BP1 gene is not a common cause of sporadic epilepsy or mental retardation.     

Frequency and distribution in East Asia of 12 mutations identified in the SLC25A13 gene of Japanese patients with citrin deficiency

Deficiency of citrin, a liver-type mitochondrial aspartate-glutamate carrier (AGC), encoded by the SLC25A13 gene on chromosome 7q21.3, causes autosomal recessive disorders: adult-onset type II citrullinemia (CTLN2) and neonatal hepatitis associated with intrahepatic cholestasis (NICCD). So far, we have described 12 SLC25A13 mutations: 11 were from Japan and one from Israel. Three mutations found in Chinese and Vietnamese patients were the same as those in Japanese patients. In the present study, we identified a novel mutation IVS6+1G>C in a Japanese CTLN2 patient and widely screened 12 SLC25A13 mutations found in Japanese patients in control individuals from East Asia to confirm our preliminary results that the carrier frequency was high in Asian populations. Mutations 851–854del and 1638–1660dup were found in all Asian countries tested, and 851–854del associated with 290-haplotype in microsatellite marker D7S1812 was especially frequent. Other mutations frequently detected were IVS11+1G>A in Japanese and Korean, S225X in Japanese, and IVS6+5G>A in Chinese populations. We found a remarkable difference in carrier rates in China (including Taiwan) between north (1/940) and south (1/48) of the Yangtze River. We detected many carriers in Chinese (64/4169 = 1/65), Japanese (20/1372 = 1/69) and Korean (22/2455 = 1/112) populations, suggesting that over 80,000 East Asians are homozygotes with two mutated SLC25A13 alleles.     

Isolation,X location and activity of the marsupial homologue of <Emphasis Type="Italic">SLC16A2</Emphasis>, an <Emphasis Type="Italic">XIST</Emphasis>-flanking gene in eutherian mammals

X chromosome inactivation (XCI) achieves dosage compensation between males and females for most X-linked genes in eutherian mammals. It is a whole-chromosome effect under the control of the XIST locus, although some genes escape inactivation. Marsupial XCI differs from the eutherian process, implying fundamental changes in the XCI mechanism during the evolution of the two lineages. There is no direct evidence for the existence of a marsupial XIST homologue. XCI has been studied for only a handful of genes in any marsupial, and none in the model kangaroo Macropus eugenii (the tammar wallaby). We have therefore studied the sequence, location and activity of a gene SLC16A2 (solute carrier, family 16, class A, member 2) that flanks XIST on the human and mouse X chromosomes. A BAC clone containing the marsupial SLC16A2 was mapped to the end of the long arm of the tammar X chromosome and used in RNA FISH experiments to determine whether one or both loci are transcribed in female cells. In male and female cells, only a single signal was found, indicating that the marsupial SLC16A2 gene is silenced on the inactivated X.     

Relation between the plasma levels of LDL-cholesterol and the expression of the early marker of inflammation long pentraxin PTX3 and the stress response gene p66<Superscript>(ShcA)</Superscript> in pacemaker-implanted patients

Abstract Our goal was to set up a pilot study to explore the possible relation between the expression of p66^(ShcA) and PTX3, two emerging regulators of stress response and inflammation processes, respectively, and the circulating levels of LDL-cholesterol (LDL), a factor implicated in the development of inflammation and oxidative-stress associated diseases such as atherosclerosis. p66^(ShcA) and PTX3 mRNA contents were determined locally, in subcutaneous adipose specimens of non-diabetic pacemakerimplanted patients, and systemically in the circulating white blood cells (WBC) obtained from the same patients. The mean of the circulating LDL levels (125 mg/dl) was chosen as a threshold to identify two groups here considered to have high (>125 mg/dl) and low (<125 mg/dl) LDL plasma levels. Our data show that PTX3 and p66^(ShcA) mRNA levels are significantly more elevated in WBCs and in adipose tissue samples of patients with high levels of LDL compared to those with low levels. Additionally, a multiple regression analysis indicates that among LDL, TG, HDL, total cholesterol, CRP, creatinine and glucose levels, the only variable significantly affecting p66^(ShcA) and PTX3 mRNA expressions either in adipose tissue or in WBCs is represented by the circulating amount of LDL. In conclusion, our results suggest a potential link between the level of LDL and the expression of two genes involved in inflammation/oxidative stress pathways, i.e., p66^(ShcA) and PTX3, thus contributing to further understand the mechanism through which LDL may mediate the pathogenesis of inflammation and oxidative-stress associated diseases such as atherosclerosis. *These two authors contributed equally to this paper